Protein molecular switches functionally couple external signals (such as ligand binding) to functionality. Molecular switches have a wide variety of potential health related applications including the regulation of gene transcription, the modulation of cell signaling pathways, targeted drug delivery, drug transport, the creation of conditionally active toxic proteins, and the creation of molecular biosensors. Despite their great potential, the creation of protein switches has not been extensively explored, in part due to the paucity of general strategies for their engineering. Using combinatorial methods that integrate biological, chemical and engineering approaches, molecular switches will be engineered by a novel strategy called 'combinatorial domain insertion' using model proteins in 'proof-of-principle' experiments. In combinatorial domain insertion, two genes are fused such that one is randomly inserted within the other. In the model system chosen, Gene A codes for a binding protein that undergoes a conformational change in the presence of a signal (e.g. ligand binding). Gene B codes for a protein to be controlled (e.g. an enzyme). From these libraries, fusion proteins will be identified that functionally couple the two domains' functions (e.g. ligand binding modulates the enzyme's activity). The functional coupling is hypothesized to result from ligand-dependent conformational/stability changes in protein A that affect the activity of protein B. Representative switches obtained will be kinetically and structurally characterized. Through the systematic analysis afforded by a combinatorial approach and the biochemical and structural characterization of the switches created, models of the mechanism of switching will be developed and tested experimentally with the goal of elucidating general) principles that can be applied to the creation of molecular switches for biomedical applications.